JOURNAL OF VIROLOGY, June 1968, p. 576-586Copyright © 1968 American Society for Microbiology

Vol. 2, No. 6Printed in U.S.A.

In Vitro Chick Embryo Cell Response to StrainMC29 Avian Leukosis Virus

D. P. BOLOGNESI, A. J. LANGLOIS, L. SVERAK, R. A. BONAR, AND J. W. BEARD

Departmenit of Surgery, Duke University Medical Ceniter, Durham, North Carolina 27706

Received for publication 29 January 1968

Strain MC29 avian leukosis (myelocytomatosis) virus induced infection, elabora-tion of virus, and morphological alteration in chick embryo cells in vitro. Virus lib-eration began within 18 hr, morphological change was detectable at about 40 hr, andthe cultures could be completely altered within 80 hr after infection. Altered cellswere about half the volume and grew at approximately twice the rate of uninfectedelements. The output of virus estimated by electron microscopy was about 140 parti-cles per cell per hr. Deoxyribonucleic acid remained constant, but ribonucleic acidincreased in both infected and control cells in adjustment to culture environment.The rates of uptake and incorporation of 3H-uridine and the incorporation of 3H-thy-midine increased in the infected cells with onset of morphological change but wereunaffected by processes of infection and virus elaboration per se. Incorporation of a"4C-amino acid mixture was slightly greater in the infected than in control cells. Thespeed of continuity of infection and massive morphological alteration constitute a

unique response to avian tumor viruses, and the system gives promise of singularvalue for detailed studies of the processes of infection and morphological change.

Strain MC29 avian leukosis virus (10) inducesneoplastic response chiefly of the myeloid hema-topoietic tissue in the intact chicken (10, 15). Incontrast to the profound myeloblastic leukemiacaused by BAI strain A (7), however, the strainMC29 disease is not notably leukemic but ischaracterized principally by neoplastic prolifera-tion of myeloid cells at the level of myelocyticdifferentiation. The cells may be distributed asdiffuse growths, myelocytomatosis, or formlocalized, solid tumors, myelocytomas. Fre-quently associated with such myeloid neoplasmsare renal adenocarcinoma and cystadenoma.Unlike the responses to avian leukosis strainspreviously studied are morphological alterationsof hepatocytes and formation of frank primarytumors of the liver (U. Heine, D. Beard, Z.Mladenov, and J. W. Beard, in prepcarcation).

Investigation of strain MC29 was recentlyextended to examination of its influence on chickembryo cells (CEC) in vitro. As observed withother avian tumor viruses (4, 14, 23-26; S.Sankaran, Ph.D Thesis, Duke University, Dur-ham, N.C., 1967), exposure of CEC to strainMC29 results in the induction of infection, theelaboration of virus, and morphological alteration(12) of the culture. With low multiplicities of virusand an agar overlay, the cells form heaps or foci(11) somewhat similar to those observed withRous sarcoma virus (RSV). Also like the response

to RSV, the cells altered by MC29 virus grow inlayers which are indicative of loss of contactinhibition. In this respect, the behavior of theMC29 cells differs from that of CEC respondingto other leukosis viruses, such as BAI A, R, andES4 strains which retain contact inhibition andform uniform monolayers (A. J. Langlois, S.Sankaran, and J. W. Beard, in preparation).A singular aspect of the response to MC29

virus, however, is the apparently exceedinglyhigh rate of morphological change which mayyield uniformly altered cultures (12) within 74hr after cell exposure in appropriate virus particlemultiplicity. This finding suggests high incidenceof cell response, and, more important, directcontinuity in a high proportion of the cells ofthose successive processes concerned with cell-virus interaction in initiation of infection, elabora-tion of virus, and alteration of cell morphology.Several strains of avian sarcoma viruses causeCEC infection and rapid morphological change,but the time required for alteration of the wholepopulation of cells appears to be greater thanwith MC29 (21, 22). Leukosis viruses other thanstrain MC29 (4, 12; S. Sankaran, Ph.D Thesis,Duke University, Durham, N.C., 1967) appar-ently induce a high incidence of infection withvirus elaboration, but altered cells appear onlyafter protracted culture periods and death ofmost of the elements initially exposed to the virus.

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In strain MC29 cultures, on the contrary, thepopulation grows progressively without notableloss of cells (12). The potential value of such asystem for investigation of the continuousprocesses of infection and conversion is apparent.

In the experiments reported here, studies weremade on the growth characteristics of CECexposed to strain MC29 (11, 12). Exploratoryinvestigations were made, also, on the kinetics ofvirus liberation and on some aspects of thephysiological processes concerned particularlywith cellular metabolism of ribonucleic acid(RNA), deoxyribonucleic acid (DNA), andprotein. Morphological and other biologicalfeatures of the system will be described in anotherreport (A. J. Langlois, S. Sankaran, and J. W.Beard, in preparation).

MATERIALS AND METHODS

Strain MC29, isolated in Bulgaria (10), was broughtto this laboratory in 1965 for further study of its etio-logical activity in the intact chicken (15). Virus fortissue culture was then obtained for passage (11) fromthe blood plasma of chickens diseased with the MC29agent. All of the experiments reported here were madewith a single pool (115-67) of tissue culture fluids. Thepooled culture fluids, passed through a 0.3-,u Milliporefilter (Millipore Corp., Bedford, Mass.) and stored at-70 C in sealed ampoules, contained 8.3 X 108 virusparticles per ml, as determined by electron microscopiccount (19).

Eggs for embryos were from a laying flock free ofresistance-inducing factor (16). Primary cell cultureswere prepared (4) from decapitated, eviscerated, andtrypsinized 10- or 11-day-old embryos seeded at 4 X106 to 10 X 106 cells per plastic petri dish (100 X 20mm; Falcon Plastics, Los Angeles, Calif.). The cellswhich attached to the dish, chiefly fibroblasts, weregrown in 10 ml of Calnek's growth medium (4) at38.5 C in a humidified 5% CO2 atmosphere. Theseprimary cultures were infected at 24 hr with 33 virusparticles per cell.

Radioisotope labels. At the time of infection and atdaily intervals thereafter, the culture medium was re-moved from all dishes, the cells were washed withphosphate-buffered saline (PBS) (6), and freshmedium was added. Each day some cultures weretreated with labeled compounds (New England Nu-clear Corp., Boston, Mass.): uridine-5-3H, 26 c/mmole; thymidine-5-methyl-3H, 16.1 c/mmole; or14C-amino acid mixture, 40 mc/milliatom of carbon.At cell harvest, the medium was sampled for radioac-tivity and virus particle count (19). The cells werewashed with PBS, removed with trypsin, sedimented,and then resuspended in PBS. A sample was taken forcell count, the cells were sedimented again, and thepellet was frozen in a dry ice-alcohol bath and storedat -20 C. In some supplementary studies of labeled-compound uptake from the medium, successive 0.2-ml samples of medium were taken from the same cul-ture dish over the interval under study.

Analysis of cells. Fractionation of labeled cells waseffected with procedures adapted from those of Scottet al. (18). The fractions were examined for radioac-tivity in a liquid scintillation spectrometer (PackardInstrument Co., Inc., Downers Grove, Ill.) and forultraviolet absorption. Acid-soluble nucleotides wereobtained by three successive extractions of cells with0.5 ml of 0.3 M HC104 at 0 to 4 C. For extraction ofRNA, the residual cell pellet was incubated for 60 minwith 0.5 ml of 1.0 M NaOH at 23 to 24 C. Protein andDNA were precipitated by addition of 0.1 ml of 6.5 MHC104 to the preparation chilled to 0 to 4 C. Thesupematant fluid from centrifugation and two pelletwash fluids of 0.5-ml volumes of 0.3 M HC104 werecombined. DNA in the insoluble material left fromRNA extraction was hydrolyzed by heating a suspen-sion of the residual pellet in 0.5 ml of 0.3 M HCl04 at90 C in a water bath for 30 min (29). The precipitatewas removed by centrifugation, and the supernatantfluid and 0.5 ml of 0.3 M HCl04 used to wash thepellet were combined for DNA analysis.Amounts of RNA in ,ug were computed by multi-

plying the difference between the optical density valuesmeasured at 260 and 320 mr,, respectively, by thevolume of the extract and the factor 32.8. This factorwas obtained from the data for the nucleotide ratiosof the whole RNA of avian myeloblasts of myelo-blastic leukemia (27) and the extinction coefficients ofnucleotides at 260 ml in acid solution (3), assumingno residual hypochromicity in the partially hydrolyzedRNA (9). An approximate estimate of acid-solublenucleotide was made with the same factor, althoughthe proportions of various nucleotides, nucleosides,and bases in the soluble pool were unknown. The cor-responding factor for calculation of DNA quantitywas 31.2, determined in like manner from nucleotideratios of chicken cell DNA (27) and extinction values(3).

Protein (13) and radioactivity were determined on'4C-amino acid-labeled cells, extracted three timeswith cold 0.3 M HC104, sedimented, and then dis-solved in 1.0 ml of 1 M NaOH.

Cell and virus-particle eniumerationz. Cell samples inPBS were mixed with equal volumes of2% glutaralde-hyde and stored at 2 to 4 C for later counts in a hemo-cytometer. Culture fluids for virus particle counts werediluted with appropriate volumes, usually 20-fold, of1%o glutaraldehyde and stored in the cold. Countswere made by electron microscopy of virus particlessedimented on agar (19).

Cell volume. Cells suspended in PBS without Ca++or Mg++ were centrifuged to equilibrium packing,2,000 X g for 30 min, at 2 to 4 C in a Van Allen bloodvolume index tube (28) with the bulb calibrated andfilled. The tubes were coated with silicone (Siliclad;Clay Adams, Inc., New York, N.Y.) to prevent stick-ing of the cells to the lower part of the bulb.

Morphological alterationi. Cells growing on coverslips placed in appropriate cultures were fixed inmethanol and stained with May-Grunwald-Giemsa.The proportion of altered cells on each cover slip wasdetermined by counts in random areas with a limited-field ocular at a magnification of 450 X. All counts,500 cells each, were made on triplicate preparations.

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RESULTSKinetics of cell growth and virus liberation.

Response of CEC to strain MC29 is characterizedin part by elaboration of virus and marked changein cell growth rate. The results of a typical experi-ment (Fig. 1) showed that the control-cell growthwas essentially constant with a population dou-bling time (PDT; 5) of about 136 hr throughoutthe 5-day study. The growth rate of the virus-treated cultures was approximately the same atfirst but then, after about 48 hr, increased ab-ruptly to a PDT of about 42 hr. In longer experi-

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FIG. 1. Characteristics of cell growth and virusliberation. Cultures in 100 X 20 mm dishes containing4 X 106 cells were exposed to 33 strain MC29 virusparticles per cell. Medium in thtese and correspondingcontrol cultures was changed at 24-hr intervals, but thecells were not transferred to new dishes during the 5daystudy. At 4, 8, atid 24 hr after each medium change, agroup ofcultures was harvestedfor cell count and virusparticle count in the culture fluid. Each control-cellpoint represents the meani of counts of four dishes, andeach MC29 cell and virus point show the mean of twocultures. Straight lines werefitted to the cell data by themethod of least squares, withl the 4- to 120-hr values forcontrol cells and the 48- to 120-hr values for MC29-treated cells. The line was drawn through the virus poisltsas previously described (2), on the assumption of loga-rithmic growth of the MC29 cells at the rate shown (42-hr PDT) and a constant rate (kv) of virus liberation(141 viriis particles per cell per hr). Apparent virusparticles in the supernatanit fluids ofthe contr-ol culturesitever reached the base lisle oj'the gralph.

ments, growth of the altered cells continued at anapproximately constant exponential rate for aslong as 2 months (12). In contrast, untreatedcontrol cells died in 2 or 3 weeks under the con-ditions of study.

Virus particles free in the medium of the virus-treated cultures first reached levels sufficient forcounting by electron microscopy (about 107particles per ml) at about 48 hr. Values of theaccumulated numbers of virus particles liberatedthereafter are charted in Fig. 1. The character-istics of the average rates of virus appearance inthe culture fluid are indicated by the close fit ofthe experimental points to the curve drawn (2) onthe assumption of a constant rate of particleliberation per cell per hour and logarithmic cellgrowth. The kv (2) calculated for the interval of48 to 120 hr in this experiment was 141 particlesper cell per hr. That this kv was not fully repre-sentative of all particles liberated has been indi-cated in other experiments. Electron micrographsof cells sectioned in situ attached to the dishes ofsimilar cultures (U. Heine, unpublished data)revealed many particles attached to the surfaceof the dish and trapped about and betweenadjacent cells. Such particles can be suspendedalso by trypsinizing the cultures.The data from experiments on the time course

of virus liberation could also be plotted, as inFig. 2, to emphasize differences in the rates ofvirus output related to time after change of culturemedium. As in other experiments, virus liberationwas demonstrable in the cultures at about 48 hr,and it appeared that the rate of liberation wasgreater in the first daily 8-hr period than in thelast 16 hr. Total numbers of virus particles perculture increased in the three intervals betweendaily changes of the medium from 48 to 120 hr ofculture (Fig. 3). The highest concentrationattained was about 2.5 x 109 particles per ml inthe interval between 96 and 120 hr. Occasionalparticles of approximately the size of virus wereevident in some fluids from cultures of normalcells.

Kinetics ofmorphological alteration. As reportedearlier (12), morphological alteration of the cellsmay be detectable within about 48 hr after ex-posure to virus and essentially complete at about3 days. For a detailed study of this feature ofresponse, virus-treated cultures prepared withthree cover slips in each dish were terminated atsuccessive intervals, and the cover slips wereremoved and stained. Counts were made of 500cells in random areas of each cover slip, and theaverages of the three values for each interval aregiven in Fig. 4. Since the percentage of morpho-logically altered cells as a function of time ap-peared to follow a sigmoid curve, the data were

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250

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FIG. 2. Rate of virus particle liberation relative to change of culture fluid at 24-hr intervals in an experiment

similar to that ofFig. 1. Cell and virus-particle counts were made on two cultures at 4, 8, 12, and 24 hr.

25 uVirus Particles,MC 29 Cultures

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Hours After InfectionFIG. 3. Concenitrations of virus particles attained on successive days inl the same cultures as those of Fig. 2. Tlle

line representing cell growth was fitted by the method of least squares, usinig the data from 0 to 48 hr andfrom 48to 120 hr separately.

first plotted on a probit scale, and a straight linewas fitted by inspection. The values from thisstraight line were then transferred to the linearscale of Fig. 4 to yield the sigmoid curve fitted

to the points. The good agreement of the experi-mental values with the curve derived in this wayindicates a normal distribution of individual cellresponse manifested by morphological alteration.

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BOLOGNESI ET AL.

14Cel Volume

Control 90

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FIG. 4. Rate of morphological con1versioni antdchanges in volume antd growth rate of CEC exposed tostrain MC29 at a muiltiplicity of 33 viru(s pacrticles percell. Estimates of percenttage of coniverted cells wereniade in triplicate, antd all other pointts are the meants ofestimates on two cultuires. Culture flhiid was changedat 24-hr intervals witholut transfer of cells.

In this study, morphological change was firstnoted at about 42 hr and was uniformly completeat about 80 hr, as estimated by this method ofexamination.

Alteration of cell volume. Inspection of theconverted cells had suggested that the alteredelements were smaller than the nontreated cells.This was investigated in the experiment justdescribed with a series of 10 cultures exposed tovirus and 10 kept as controls. Medium waschanged at 24-hr intervals, and two cultures foreach group, virus-treated and not treated, weretrypsinized for cell count and estimate of cellvolume. As suspected (Fig. 4), there was a markeddecline in the mean volume of the virus-treatedcells which was closely related chronologically tothe increase in the rate of cell growth. Controlcells and those exposed to virus exhibited a PDTof about 112 hr for about 24 hr after introductionof virus, but thereafter, in the 4-day period, thePDT of the infected cultures decreased to about54 hr. In this interval, decline in cell volumewas inversely related to increase in cell number.Observations were extended for 24 days withcultures of cells derived from the same embryostock as those used in the study of Fig. 4, buttrypsinized and transferred as necessary. In thisseries (Fig. 5), the mean volume of the infected

cells continued to decline for 9 days, and at thistime the value was 4.5 mm3/106 cells as comparedto 10.2 mm3/106 control cells. Little furtherchange occurred in the remaining 15 days ofobservation.

Clanges in nucleic acid and protein. The markedchanges in cell growth rate, size, and morphologyindicated that substantial changes in cell metab-olism were occurring. It was of interest to examinethe time course of such changes, to detect thosewhich might be associated with the various eventsof infection, virus elaboration, and cell trans-formation. Exploratory experiments have revealedchanges associated with morphological alterationbut not with the processes of virus infection.

Analyses of nucleic acids and related com-ponents in three experiments yielded consistentresults. Some of the data obtained in studies onthe cultures of Fig. 1 are shown in Fig. 6 and 7.Throughout the 5-day study, DNA amounts re-mained essentially constant at the average levelof about 3.3 pg/106 cells without evident differ-ences between infected and normal cells. RNAcontent, in contrast to DNA, showed markedchange in both control and virus-exposed cellsduring the course of the study. RNA amount, incomparison with that of DNA (Fig. 7), increasedto approximately 70%>/-, above the initial value in3 days and then declined somewhat. The closeparallelism of the data from the virus-treated andcontrol cultures indicated that the changes were

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related to the processes of adaptation of the cellsto conditions in vitro without any influence ofinfection or morphological alteration.

Acid-soluble nucleotides, however, behaveddifferently. As in the case of RNA, the nucleotidepool increased sharply in both control and virus-treated cells for about 3 days (Fig. 6). In theMC29 cells, however, there was a marked declinebetween 3 and 5 days, whereas, in contrast, thehigh level was maintained in the control cells. Thefraction containing nucleotides, which was notanalyzed further, presumably contained nucleo-sides and bases as well. Nevertheless, nucleotidesprobably constituted the bulk of the total materialmeasured by ultraviolet absorption (17).

Incorporation of 3H-thymidine into DNA. Thestudies showed no increase in the cell content ofDNA in either control or virus-treated culturesduring the 5-day period of examination. Toinvestigate further the metabolism of DNA,cultures were prepared from the same materialand at the same time as those of Fig. 1 and 6, asindicated in Fig. 7. The rates of 3H-thymidineincorporation increased slightly in both controland virus-treated cultures for approximately 2days. In the interval between about 2 and 3 days,however, the specific activity of the DNA rosesharply and then leveled off after 80 hr. There was

48 72Hours After Infection

FIG. 6. Acid-soluble nucleotide and RNA concen-trations in MC29-treated and control cells in terms ofthe constant average value of 3.3 pg of DNA/J06 cells.RNA and DNA analyses were made on the same cul-tures yielding the data of Fig. 1. Each point is the aver-

age of two single determinations on the respectiveduplicate virus-treated and control cultures.

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FIG. 7. Incorporationi of 3H-thymidil2e ilito DNA ofvirus-treated and control cultures. The cultures were

prepared in duplicate with the same materials and at thesame time as those of Fig. 1 and 6. Culture fluids were

changed, and the cells were exposed to 2 pc (per ml) oftritiated 5-methyl thymidine for 8 hr. Values for specificradioactivity are the average of the data from two cul-tures.

but slight increase in the specific activity of theDNA of the control cultures in the 5-day period.At 80 and 104 hr, the specific activity of MC29cell DNA was approximately 2.7-fold that of thecontrol cells, which was in fair agreement with therelative rates of growth in the respective cultures(Fig. 1).

Disposition of 3H-uridine added to cultures.Analyses of 3H-uridine removal from the culturemedium revealed major differences between thebehavior of the control and that of the virus-treated cultures. Figure 8 discloses the results ofstudies with the same cultures yielding the resultsof Fig. 2 and 3. Measurements of radioactivityremaining in the culture fluid at intervals afteraddition of 3H-uridine on the different daysshowed a nearly constant rate of label disap-pearance in the control cultures. Beginning onthe 3rd day, however, there was a much increasedinitial rate of disappearance of label in the virus-treated cultures. This initial rate was then pro-gressively greater from 3 to 5 days, and the timerequired for maximal removal of label declinedfrom 12 to 8 to 4 hr. Nevertheless, the proportionof label disappearing from the medium on anyday did not exceed 50%O of that added in thisstudy. By centrifugation of the used medium, it

,

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HOURS AFTER ADDITION OF LABELFIG. 8. Analysis of the disappearance of tritium label from the extracellular medium and the incorporation of

the label in the cellular nucleotide pool and RNA. The cultures were the same as those of Fig. 2 and 3 consistingof pairs, and the exposure multiplicity was 33 virus particles per cell. Medium was changed each day without celltransfer. Pairs of conztrol and virus cultures were treated each day with I j,c (per ml) of3H-uridine and terminated2, 4, 8, 12, and 24 hr later. Samples of the media were analyzedfor radioactivity, and uptake (upper curves) wascalculated as the difference between the amount added and that found. The middle series of curves represents theradioactivity of the acid-soluble nucleotides extracted from the cells, and the lower series shows RNA radioactivity.Each point is the average ofmeasurements on duplicate cultures. Symbols: O, normal cells; 0, virus-infected cells.

was determined that less than 5% of the residualradioactivity could be accounted for in virusparticles or sedimentable cell debris.Marked differences were likewise observed in

the specific radioactivity of the nucleotide poolsof normal and MC29 cells (Fig. 8). In the controlcultures, the specific activity of the pool increasedthroughout the initial 24-hr period of study. Be-ginning with the 2nd day and continuing through-out the remaining 4 days, however, the patternchanged somewhat, the activity increasing until8 hr and then declining slightly. The virus-con-taining cultures behaved like the controls for thefirst 2 days in this respect, but later the increasingrate of label removal from the medium was ac-

companied by an increasingly rapid rise and fallof specific activity of the nucleotide pool, the fallbeginning with the cessation of uptake. The termspecific activity is applied somewhat loosely to theacid-soluble nucleotide pool, since the values arebased not on the amount of uridine or uracilcompounds alone, but on the total of all nucleo-tides, nucleosides, and bases, as measured byultraviolet absorption.The specific activity of RNA in the infected

cells also began to exceed that of control cellRNAby the end of the 2nd day (Fig. 8). The initial rateof labeling of RNA from the 3rd through the 5thday was about two to three times more rapid inthe infected cells than in the controls, but the

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maximal level of specific activity reached wasprogressively lower. In relation to the cessation ofuptake of labeled uridine and the rapid declinein the specific activity of the acid-soluble pool,which occurred at about the same time, the pointsat which specific activity of the RNA stoppedincreasing were delayed by about 4 hr.

Similar experiments, in which 3H-cytidine wasthe labeled precursor or in which different cellconcentrations were used, yielded qualitativelysimilar patterns of isotope uptake from themedium, as well as labeling of the acid-solublepool and of RNA. The changes in the specificradioactivity of the RNA are dependent on severalfactors, including the rate of RNA synthesis andthe specific activity of the uridine nucleotide pool.The latter depends upon the rates of uptake of3H-uridine from the medium and de novo synthe-sis of uridine, as well as incorporation of the 3H-uridine into RNA and metabolism through otherpathways. Further studies on the detailed com-position of the nucleotide pool, as well as itsmetabolism, will be necessary for a better under-standing of the marked differences betweencontrol and altered cells.

Additional studies were made to claiify thestriking apparent limitations in uptake of 3H-uridine from the medium by the MC29 cultures(Fig. 8). The amount of 3H-uridine made availableto identical cultures of fully altered cells made nodifference in the proportion of label removed fromthe medium. When a portion of used medium wasmixed with fresh unlabeled medium and offered tosimilar cell cultures, almost none of the residualradioactivity was taken up. Similar results wereobtained when a control culture was used con-taining the same number of cells as the MC29culture. It appears, therefore, that the radioac-tivity remaining in the medium probably repre-sented metabolic products of a portion of uridinewhich was not utilized for RNA synthesis. Aboutone-half of the residual radioactivity of the spentmedium was volatile (40 C, atmospheric pressure)and probably was largely in the form of tritiatedwater.

Uptake and incorporation of '4C-labeled aminoacids. Little difference between the MC29 andcontrol cultures was noted in the quantitative orqualitative aspects of the uptake of a "4C-labeledamino acid mixture. The maximal amount oflabel removed from the medium was about 20%during any day in the period under study. Thespecific activity of the protein was similar in thecontrol and virus-treated cells for the first 32 hr,but divergence was observed at 48 hr with theprotein of the MC29 cells showing a greater

incorporation of label. This time correspondedto the beginning of increased growth.

DIscussioN

Investigation of response of primary CECcultures to MC29 strain avian leukosis virus hasdisclosed cultural changes of two principal kinds:(i) behavior of the virus-exposed cells parallelingthat of the control cultures related presumably toenvironmental effects on all cells newly introducedto in vitro conditions; and (ii) activities of thevirus-exposed cells differing from those of thecontrols and thus related to virus-cell interaction.A variety of minor changes occurred in all

cultures in the first few days of each study. Itshould be emphasized that the experiments weremade with primary cultures exposed to virusonly 24 hr after preparation from the embryo.In the initial interval of approximately 24 to 48hr after introduction of virus into the cultures,there was slow growth of both control and virus-treated cultures at rates of about 130-hr PDT.This was continued by the control cells for the5-day study period in contrast to the change torapid growth of virus-exposed cells occurring atabout 24 to 48 hr. In both infected and controlcultures, the amount of DNA per cell remainedessentially constant during the 5-day study, whilesome increase in RNA per cell was observed inboth cultures.Among the various aspects of response to

MC29 strain, the most striking was the alterationin cell morphology which differed markedly in itsrapidity and uniformity from that of CEC re-sponding to other avian leukosis virus strains(4, 12; S. Sankaran, Ph.D Thesis, Duke Univer-sity, Durham, N.C.; A. J. Langlois, S. Sankaran,and J. W. Beard, in preparation). A characteristicfeature of the morphological response to exposureto a sufficient number of virus particles was thespeed of onset and, of more importance, the speedof alteration of most or all of the cells in thecultures. Under suitable conditions, such as thosein the experiment of Fig. 4, alteration began atabout 40 hr, and the increase in percentage ofaltered cells to about 80 hr followed a sigmoidcurve, indicating a normal distribution of re-sponse of individual cells in the total population.The relative rapidity and completeness ofmorphological alteration of the culture, requiringan interval corresponding only to about one PDT,suggested that most or all of the cells of the culturewere infected initiallv and were altered as a directconsequence of that infection. For additionalevidence on this point, a few experiments weredone with cells exposed to virus, washed, andplated immediately on irradiated feeder CEC

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(A. J. Langlois and D. P. Bolognesi, unpublisheddata). Although the plating efficiency was only 8to 10%, every clone of about 1,000 examinedconsisted entirely of altered cells. Further studyis necessary, however, to establish unequivocallythe proportion of cells initially exposed to viruswhich become infected and respond directly bymorphological alteration.

It was notable that all other aspects of changenot paralleling the behavior of the control cultureswere remarkably closely related to the onset ofmorphological alteration. Most noticeable wasthe decrease in cell size. That the change in sizewas not fully related to other aspects of alterationwas indicated by continued decline in cell volumefor several days after the other characteristicchanges in cell morphology were apparentlycomplete. Decrease in size was considerable,reaching levels less than 50% of the volume of thecontrol cells. There were also minor changes inthe size of the control cells.

Attending these morphological alterations wasthe simultaneous major increase in growth ratecorresponding to a PDT of approximately 30to 50 hr, as compared with about 130 hr for thecontrol cells. The higher rate was attainedabruptly but continued at a fairly constant levelof exponential growth for as long as 2 months(12), whereas the control cells did not survivefor more than 2 to 3 weeks under the conditionsof the experiments.

Concurrent with the increase in growth ratewere increases in the rates of DNA and RNAsynthesis and changes in uridine uptake andspecific activity of the nucleotide pool. There wasa smaller increase in the rate of protein synthesis.Amounts of DNA and RNA per cell remainedessentially constant.

Virus liberated and suspended in the culturemedium reached concentrations sufficient forelectron microscopic count at about 48 hr. There-after, the rate of output was essentially constantat the level (Fig. 1) of about 140 particles per cellper hr. That this did not represent the total outputhas been evident from electron micrographs show-ing virus lying among the cells after withdrawal ofthe fluid and by examination of fluids fromtrypsinized cultures. Although attainment of theparticle concentration requisite for electronmicroscopic study coincided approximately withbeginning morphological change, it is unlikelythat the high output of virus was related to cellalteration. Virus is liberated at similar intervalsafter exposure to BAI A, R, ES4, and RPL 12leukosis virus strains long before morphologicalalteration is recognizable (12; A. J. Langlois, S.Sankaran, and J. W. Beard, in preparation). The

relationship in MC29 cultures would appear to beentirely fortuitious and caused only by the speedof morphological change. Preliminary studies(A. J. Langlois and D. P. Bolognesi, unpublisheddata) indicated that measurable infectious MC29virus began to appear within 18 hr after exposureto virus, which was about 30 hr before the onset ofalteration.The rate of virus output by the MC29 cultures

was in the same range as that observed with CECcultures infected with the other leukosis viruses.Rates as high as 500 to 800 particles per cell per hrhave been observed (12) in occasional experimentswith strain R and as low as 2 to 10 particles percell per hr with strains ES4 and RPL 12. The rateof virus liberation appeared to be strongly de-pendent on the particular embryo from which thehost cells were derived, as well as on the infectingvirus strain. Variation in virus-synthesizingcapacity among cells from different birds had beenobserved earlier in studies on infection of bonemarrow by BAI strain A (1).The response of CEC to strain MC29 is of par-

ticular interest, on the one hand, because of therapid continuity of the processes of infection andmorphological alteration. In addition, the be-havior of the cultures is the more remarkable,since no other avian leukosis strain has been ob-served to induce changes in this manner in chickembryo cell cultures. Although the strain MC29may be complex, none of the studies thus far,either in the chicken or in tissue culture, has givenevidence of a sarcoma virus component. It is thusevident that the influence of this virus, responsiblefor neoplastic response principally of hemato-poietic tissue in the intact host, does not differ inprinciple from that of sarcoma-inducing agentsunder in vitro conditions. The effects of MC29 onCEC exhibited similarities to those exerted byRSV, but differences were also evident. MC29-altered cells were smaller and grew much morerapidly than control cells, in contrast to RSV-altered cells which may be larger and grow moreslowly than uninfected cells (8, 20-22). The rapidmorphological alteration of all the cells in a cul-ture by MC29 occurred within an interval corre-sponding essentially to a single PDT.

Exploratory biochemical studies have revealeddifferences between the metabolism of MC29-altered cells and that of the control cells. Analo-gous investigations with RSV systems haveyielded conflicting results (8, 21) which precludecomprehensive comparison of the respective ac-tivities of the RSV and MC29 cultures with pres-ent information. The studies of DNA and RNAin MC29-infected cells served to outline the rela-tionship of nucleic acid metabolism to morpho-

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logical alteration, to locate the changes in time,and, thus, to serve as a basis for more detailedinvestigations of this important phenomenon.The fact that no differences in nucleic acid amountor synthesis were seen in relation to the establish-ment of infection or particle liberation was to beexpected. The amount of RNA and protein re-quired for virus production is extremely smallcompared to that used in continuing cell growth(30).

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service grant C-4572; by the Annie Mabel SherrisMemorial Grant for Cancer Research from the Ameri-can Cancer Society, Inc.; and by the Dorothy BeardResearch Fund.The chick embryos used in this work were from

White Leghorn eggs obtained from Roy Luginbuhl,University of Connecticut, through the ResearchResource Program of the National Cancer Institute.

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2. Beaudreau, G. S., C. Becker, T. Stim, A. M. Wall-bank, and J. W. Beard, 1960. Virus of avianmyeloblastosis. XVI. Kinetics of cell growthand liberation of virus in cultures of myelo-blasts. Natl. Cancer Inst. Monograph 4:167-187.

3. Beaven, G. H., E. R. Holiday, and E. A. Johnson.1955. Optical properties of nucleic acids andtheir components, p. 493-553. In E. Chargaffand J. N. Davidson [ed.], The nucleic acids,vol. 1. Academic Press, Inc., New York.

4. Calnek, B. W. 1964. Morphological alteration ofRIF-infected chick embryo fibroblasts. Natl.Cancer Inst. Monograph 17:425-447.

5. Committee on Terminology, The Tissue CultureAssociation. 1967. Proposed usage of animaltissue culture terms. J. Nati. Cancer Inst. 38:607-611.

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8. Golde, A. 1962. Chemical changes in chick em-bryo cells infected with Rous sarcoma virusin vitro. Virology 16:9-20.

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11. Langlois, A. J., and J. W. Beard. 1967. Converted-cell focus formation in culture by strain MC29avian leukosis virus. Proc. Soc. Exptl. Biol.Med. 126:718-722.

12. Langlois, A. J., S. Sankaran, P.-H. L. Hsuing,and J. W. Beard. 1967. Massive direct conver-sion of chick embryo cells by strain MC29 avianleukosis virus. J. Virol. 1:1082-1084.

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14. Manaker, R. A., and V. Groupe. 1956. Discretefoci of altered chicken embryo cells associatedwith Rous sarcoma virus in tissue culture.Virology 2:838-840.

15. Mladenov, Z., U. Heine, D. Beard, and J. W.Beard. 1967. Strain MC29 avian leukosis virus.Myelocytoma, endothelioma and renal growths:Pathomorphological and ultrastructural as-pects. J. Natl. Cancer Inst. 38:251-285.

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